Methods: A Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33&nbsp;Hz with 20% elongation for 0&nbsp;h, 6&nbsp;h or 24&nbsp;h. The cells were stretched alone or pre-treated with Cytochalasin D. The redistribution of the actin cytoskeleton was evaluated using phalloidin immunofluorescence staining. The protein expression levels of IL-8 and JNK in the RPE cells were determined via Western blotting.

Results: The cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24&nbsp;h, phalloidin staining revealed an unclear and irregular actin cytoskeleton. In all Cytochalasin D-treated cells, the shrinkage and disruption of the cytoskeletal structure was observed regardless of mechanical stress. The stimulation of the RPE cells with cyclic stretch alone did not induce a significant increase in IL-8 expression and JNK phosphorylation levels, which were similar to those of the control groups. After pre-treatment with Cytochalasin D alone, IL-8 expression and JNK phosphorylation levels were not significantly different at 6&nbsp;h but were significantly increased by approximately 1.2-fold (1.18&nbsp;&plusmn;&nbsp;0.05; P&lt;0.01) and 3.0-fold (3.01&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 24&nbsp;h, respectively. After the pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch, IL-8 expression and JNK phosphorylation levels increased by approximately 1.3-fold (1.31&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) and 1.3-fold (1.31&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 6&nbsp;h, respectively, and by 1.7-fold (1.69&nbsp;&plusmn;&nbsp;0.06; P&lt;0.01) and 3.2-fold (3.21&nbsp;&plusmn;&nbsp;0.12; P&lt;0.01) at 24&nbsp;h, respectively.

Conclusions: This study demonstrates that disruption of actin polymerization by cytochalasin D and mechanical stretch upregulates interleukin-8 expression and JNK phosphorylation levels in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in the pro-inflammatory processes in RPE cells.

Fig1: Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments

Mentions:
The effect of mechanical stretch and Cytochalasin D on the actin cytoskeleton was assessed via fluorescence confocal microscopy using phalloidin-Rhodamine staining on ARPE-19 cells. The cells in the control groups displayed abundant and uniform phalloidin staining (Fig. 1a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (P<0.05; n = 3) (Fig. 1f, Fig. 1m ). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (Fig. 1g-l, Fig. 1m).Fig. 1

Fig1: Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments

Mentions:
The effect of mechanical stretch and Cytochalasin D on the actin cytoskeleton was assessed via fluorescence confocal microscopy using phalloidin-Rhodamine staining on ARPE-19 cells. The cells in the control groups displayed abundant and uniform phalloidin staining (Fig. 1a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (P<0.05; n = 3) (Fig. 1f, Fig. 1m ). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (Fig. 1g-l, Fig. 1m).Fig. 1

Methods: A Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33&nbsp;Hz with 20% elongation for 0&nbsp;h, 6&nbsp;h or 24&nbsp;h. The cells were stretched alone or pre-treated with Cytochalasin D. The redistribution of the actin cytoskeleton was evaluated using phalloidin immunofluorescence staining. The protein expression levels of IL-8 and JNK in the RPE cells were determined via Western blotting.

Results: The cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24&nbsp;h, phalloidin staining revealed an unclear and irregular actin cytoskeleton. In all Cytochalasin D-treated cells, the shrinkage and disruption of the cytoskeletal structure was observed regardless of mechanical stress. The stimulation of the RPE cells with cyclic stretch alone did not induce a significant increase in IL-8 expression and JNK phosphorylation levels, which were similar to those of the control groups. After pre-treatment with Cytochalasin D alone, IL-8 expression and JNK phosphorylation levels were not significantly different at 6&nbsp;h but were significantly increased by approximately 1.2-fold (1.18&nbsp;&plusmn;&nbsp;0.05; P&lt;0.01) and 3.0-fold (3.01&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 24&nbsp;h, respectively. After the pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch, IL-8 expression and JNK phosphorylation levels increased by approximately 1.3-fold (1.31&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) and 1.3-fold (1.31&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 6&nbsp;h, respectively, and by 1.7-fold (1.69&nbsp;&plusmn;&nbsp;0.06; P&lt;0.01) and 3.2-fold (3.21&nbsp;&plusmn;&nbsp;0.12; P&lt;0.01) at 24&nbsp;h, respectively.

Conclusions: This study demonstrates that disruption of actin polymerization by cytochalasin D and mechanical stretch upregulates interleukin-8 expression and JNK phosphorylation levels in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in the pro-inflammatory processes in RPE cells.